Polyclonal antibody libraries to human colorectal cancer

The long-term goal of this work is to develop a treatment for colorectal cancer that utilizes tumor-reactive recombinant polyclonal antibody libraries (PCALs), standardized mixtures of polyclonal antibodies for which the encoding genes are available. Unlike monoclonal antibodies, PCALs would be directed against many different antigens on the cancer cell and thereby would reduce the likelihood of emergence of cancer cell variants that are no longer recognized by the antibodies.; As a first step in producing a PCAL to colorectal cancer, a combinatorial Fab phage display library was generated from the antibody variable region genes of mice immunized with three human colorectal cancer cell lines. The library was selected on the colorectal cancer cells by two different methods. In selection method 1, the Fab phage display library was selected in two successive rounds on a suspension of formaldehyde-fixed human colorectal cancer cells. In selection method 2, the library was selected in one round on a suspension of human colorectal cancer cells, in native form, using density gradient centrifugation to separate cell-bound and free phage. Analysis of the selected libraries, by ELISA and DNA fingerprinting, showed that a first round of selection, by either method, yielded a high percentage of antigen-reactive clones (54% and 91% for methods 1 and 2, respectively) and a high degree of polyclonality. In method 1, the second round-selected library showed 94% antigen-reactive clones but more limited diversity compared to the first round selected library.; To test the hypothesis that a PCAL could be produced that preferentially recognizes cancer cells over normal human cells with a high signal to noise ratio, the round 1 libraries selected by the two methods were combined and absorbed on normal human blood cells. Analysis of the absorbed library by ELISA, DNA fingerprinting, and binding profile, showed that the absorbed library, while still diverse, has a 2–10 fold higher binding ratio to colorectal cancer cells over blood cells compared to an unabsorbed library. These results demonstrate that polyclonal Fab phage display libraries can be generated, with high reactivity to human colorectal cancer cells compared with reactivity to normal human blood cells.